DESCRIPTION: Nitric oxide (NO), a gaseous free radical generated by the NO synthase (NOS) group of enzymes, regulates proliferation and function of osteoblasts. The objectives of this proposal are to determine the effect of mechanical stimulation on intracellular distribution, modification and enzyme activity of endothelial NOS (eNOS) and determine whether mechanically-induced regulation of osteoblast-associated genes is dependent upon NO release. Furthermore, the mechanism by which mechanically-inducible genes in osteoblasts potentially mediate osteoclast activity will be explored, and an in vivo model used to determine localization and level of expression of mechanically induced gene product. The study is based on the hypotheses that a response of osteoblasts to mechanical stimulation involves activation of constitutive NOS and regulation of NOS gene expression. The second hypothesis is that mechanically-induced NO release by osteoblasts influences osteoclast development and function. The majority of the proposed experiments will utilize the immortalized mouse osteoblastic cell line MC3P3-E1. Cell monolayers will be mechanically stimulated by application of fluid shear stress and by inducing strain by stretching cells on flexible membranes. Post-translational modifications of eNOS by mechanical treatment of osteoblasts will be examined by determining whether the enzyme is palmitoylated and/or myristoylated. Subsequently, the changes in intracellular localization of the modified eNOS protein will be examined by determining the presence of eNOS in Triton-insoluble and Triton-soluble cellular fractions, and by observing the effect of mechanical treatment on eNOS intracellular distribution by immunofluorescence microscopy. The responses to mechanical stimulation of two earlier identified mechanosensitive genes (colony-stimulating factor-I and insulin-like growth factor-I) and two additional target genes (tumor necrosis factor and RANKL) will be assessed using RT-PCR, Northern blotting for mRNA and Western blotting for proteins. Dependence of mechanical responses of these genes upon NO release will be determined and the mechanism by which their protein products influence osteoclast activity will be explored. In addition, the possibility of a direct effect of osteoblastic NO on the formation and function of osteoclasts will be assessed in osteoclast-like cells RAW. Finally, localization and level of expression of NOS isoforms and other targeted osteoblast-associated genes will be evaluated in vivo, using dento-alveolar complex of mouse incisors. In addition to research, Dr. Danciu?s training plan includes 10 percent teaching and 10 percent clinical activities throughout the fellowship period. The candidate?s teaching time to both predoctoral and postdoctoral students would involve oral as well as general pathology, and she is also planning to take the Clinical Specialty Board Examination during the first year of the fellowship.